Enterotoxins : are the product of the metabolism of certain strains of cells or bacilli that have a degree of toxicity to the human body. Enterotoxins are unbranched single-chain proteins , generally composed of relatively large amounts of lysine , tyrosine , glutamic acid , and aspartic acid . It is usually not heat-labile , that is, resistant to heat, which means that many of the usual hygienic measures are not effective. As a general rule, they are sometimes responsible for food poisoning .
Summary
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- 1 Staphylococcal enterotoxins
- 2 Classification
- 3 Pathogenicity
- 4 Methods for evaluating presumptive toxigenic foods
- 5 Methods of obtaining and purifying staphylococcal toxins
- 6 Obtaining antisera
- 7 Methods for detecting staphylococcal toxins
- 8 Source
Staphylococcal enterotoxins
Enterotoxins are unbranched single-chain proteins made up of relatively large amounts of lysine, tyrosine, aspartic acid, and glutamic acid. Molecular weights range from 28,000 to 35,000 daltons, are soluble in water and have high thermostability.
The production of enterotoxins by Staphylococcus aureus strains is affected by the quality of the nutrients and the pH of the substrate, the temperature, the atmosphere, sodium chloride, other chemical compounds, and competing microorganisms. The foods most associated with outbreaks of food poisoning are cold salads, poultry products (mainly chicken salad), creamy sweets, dairy products (mainly cheeses) and ham, especially cooked or foods made with it.
Classification
Staphylococcal enterotoxins are produced by very specific strains; however, one of them is capable of synthesizing more than one enterotoxin serotype. Currently they are differentiated by their serological activity not less than 7 enterotoxins that are designated A, B, C1, C2, C3, D and E. The enterotoxin of serotype A is the one that most frequently appears in food poisoning outbreaks and is followed by in decreasing order those of serotypes C1, B, D and E.
The originally described enterotoxin F has been identified as the toxin-producing toxic shock syndrome (TSST-1) in women and occasionally in men. It is a multisystemic disease characterized by the sudden onset of fever, vomiting and diarrhea, hypotension, conjunctival redness, “strawberry tongue” and rash (with subsequent desquamation).
Current research indicates that the production of TSST-1, made by S. aureus strains, maintain a direct relationship with some toxigenic serotypes that cause outbreaks of food poisoning, in studies that have been carried out on strains isolated in humans, food and domestic animals. .1-2
Pathogenicity
When food contaminated with staphylococcal enterotoxins is ingested by humans, symptoms manifest abruptly 2 to 6 h later. Nausea, vomiting, general malaise, diarrhea and, in severe cases, prostration, cramps and shock due to a sudden drop in blood pressure appear.
Symptoms usually last less than 24 hours and the disease is rarely fatal, although fluid loss due to vomiting and diarrhea may occasionally need to be replaced. Furthermore, the growth of enterotoxin-producing staphylococcal strains has been found in a patient with pseudomembranous enterocolitis after treatment with broad-spectrum antibiotics.5 The disease manifests itself as abdominal cramps, severe diarrhea, and dehydration.
The presence of enterotoxigenic staphylococcal strains resistant to isolated drugs in pure cultures of these patients, intestinal necrosis due to the secretion of enterotoxins and certain clinical manifestations distinguish the disease from food poisoning.
The emetic receptor sites for toxins that cause poisoning are the abdominal viscera, where the sensory stimulus reaches the vomiting center via the parasympathetic (vagal) and sympathetic afferent pathways.
Enterotoxin-induced diarrhea has been attributed to decreased absorption of water from the lumen of the intestine and a simultaneous increase in secretion of water and mineral salts from the intestinal glands in a mechanism mediated by 3-5 AMP. Values of less than 1.0 mg of enterotoxin in 100 g of food are considered sufficient to induce clinical symptoms in susceptible individuals.
Methods for evaluating presumptive toxigenic foods
Contaminated foods often have high levels of enterotoxigenic staphylococci (? 106 CFU / g). The production of coagulase and thermostable deoxyribonuclease (thermonuclease) is used as the basis for the causal diagnosis. Detection of this enzyme, which is a globular protein made up of single-chain polypeptides, has the property of breaking the helical structure of DNA between purine or pyrimidine bases and phosphoric acid.
The presence of a pinkish halo on the surface of a gelled medium indicates its presence in the food. The thermostability of this enzyme is its only association with the growth of the microorganism and indicates the possible appearance of enterotoxins in food.4 Bennet5 considers that the production of this protein can be found with equal frequency in enterotoxigenic and non-toxigenic strains. However, in food poisoning outbreaks in 13 Cuban provinces, Bécquer found a high percentage of positivity (96%) between the toxigenic strains and the presence of the enzyme, which is in agreement with the findings of other researchers.
Methods of obtaining and purifying staphylococcal toxins
The first investigations began in the early 1960s and different toxigenic serotypes were purified from enterotoxin-producing strains, using various combinations of ion exchange chromatography and gel filtration, the first purifications took a long time, and were carried out in 4 or 5 stages, but a recovered bass was obtained.
In studies carried out by Bécquer et al. In obtaining and purifying serotype B enterotoxin, 3 purification steps were used, based on the use of Amberlite IRC-50 (H), carboxymethyl cellulose (CM-321) and Sephadex G-75. , and a total recovery of 15% and a high degree of purity were obtained. More encouraging results were subsequently presented in the isolation and purification of enterotoxin E, 10 when 89.72 mg of enterotoxin were obtained and a total recovery of 17.67%.
Staphylococcal toxins have been purified in 1 and 2 steps. The first step uses triazine red affinity chromatography for the purification of enterotoxin A, and the second consists of an ion exchange on carboxymethyl cellulose and a chromatic approach for enterotoxins of serotypes A, B and C where they demonstrate purity from 72 to 92%, according to a polyacrylamide densitogram.
Obtaining antisera
Some trials on obtaining antisera for staphylococcal enterotoxins have been described. The use of immunological methods depends on the availability of the specific antisera; most have been obtained in rabbits, although other animals are also used. The enterotoxin used as an antigen must be of a high purity to minimize the presence of cross-reactions with other toxins and antigens.
Initially, a long series of inoculations was used to immunize rabbits with different injectable doses of enterotoxoids followed by doses of toxins; subsequently the inoculations began to be used in very small amounts, between 2 and 5 mg, then they were increased between 1 and 4 mg using different immunization schedules; two of these schemes required very small amounts of toxins: 167 and 220 µg.
To obtain antisera of different enterotoxigenic serotypes, the most widely used immunization scheme uses rabbits that are inoculated with intervals of 8 to 10 days, with 5, 10, 25, 50, 100, 250, and 500 mg of enterotoxins to finish with 2 or 3 inoculations of 100 mg; 112 days are used in this scheme.
In studies carried out by Bécquer et al. where less than 64 days with an interval of 7 days were used and higher concentrations of toxins (10, 50, 200, 1,000, 2,000 and 4,000 mg) suspended in complete Freunds’ adjuvant (CFa) were inoculated, Acceptable antisera titers of 1:16 and 1:32 to enterotoxins A and C2 respectively, and saved half the time spent on other schemes.
Methods for detecting staphylococcal toxins
The amount of toxins that may be present in a food may be minimal (50 ng of food). However, in foods that cause outbreaks of food poisoning, their presence ranges from 1 to 5 mg / g of food.
The purification of toxins and the demonstration of their antigenicity have allowed the use of serological methods for the detection of these in culture filtrates. The serological methods used initially were simple diffusion (Oudin’s tube); simple radial immunodiffusion, which involves the diffusion of the toxin within a gel that contains homologous antibody obtaining a visible precipitation halo, which can be used to quantify the toxin; double radial immunodiffusion (Ouchterlony), a semiquantitative method that can detect toxins between 5 and 10 mg / mL; the Optimum Sensitivity Plate (OSP), which is used for screening bacterial cultures, and the most commonly used, the foil method, which has a sensitivity between 0.01 and 0.1 mg / mL and only requires 20 mL shows.
To detect such levels of toxins in food and bacteria culture filtrates requires a long period in terms of concentration and extraction of food extracts.
Electroimmunodiffusion and hemagglutination, which have a sensitivity between 1 and 10 ng, have also been used, but non-specific reactions have been observed with some of the components of various foods.
Initially, a method was used in our laboratory by which the toxin was isolated by concentration of the strain, culture filtering (cellophane technique on agar) and finally the toxin was detected by gel diffusion.
These and other techniques have made it possible to study outbreaks of food poisoning and to investigate the human origin of the isolated toxigenic strains by means of phage typing tests. Currently, an analysis method has been introduced in our laboratory that allows toxins to be detected directly in food by biochemical and immunological methods (double diffusion), which has been revealed in a work called Staphylococcus aureus proliferation , a relationship between its activity thermonuclease and enterotoxin production by Bécquer A, et al. (unpublished). In this determination there is no need to concentrate the feed, reproducibility is better and much less time is required.
Radioimmunoassay (RIA) is one of the novel methods of enterotoxin analysis that can detect levels between 1 and 10 ng / g in a time that varies between 3 and 4 h, but requires expensive equipment and is dangerous to health by the manipulation of radioactive elements. The detection of the toxin by the immunoenzymatic method (ELISA), which detects the same concentrations with similar time consumption as the RIA, is the most feasible in many laboratories due to the multiplicity of determinations that can be carried out, although sometimes it has some drawbacks in regarding the components of some foods, which sometimes interfere and produce losses regarding specificity and sensitivity.
The passive reversal of hemagglutination with latex (RPLA) is based on the fact that particles of this product sensitized with purified antienterotoxins can agglutinate with the presence of the homologous enterotoxin; This test is very sensitive because it detects amounts between 1 and 2 ng / mL and requires a time of 24 h for its execution. Currently, high-density latex particles are used that are capable of detecting 0.5 ng / mL of toxins in 3 hours.
Recombinant DNA offers many possibilities for the development of new analytical techniques in order to detect any contaminating microorganisms in food, so in the near future methods by this and other routes will probably be applied18-22 that will allow a more efficient and faster diagnosis of staphylococcal enterotoxins.
